Enveloped viruses enter and infect cells by fusion of viral and cellular membranes. The fusion step is a potential target for therapeutic intervention, just as it is the taret of inhibition by some neutralizing antibodies. We propose four aims, (1) From the structure of the dengue virus envelope protein in its trimeric, postfusion conformation, we have developed a high throughput screen for small molecule inhibitors of viral entry. We will further characterize a set of compounds we have recently identified (for dengue virus type 2) that inhibit viral infectivity in culture, and we will screen new libraries to identify additional compounds scaffolds. We will examine the extent to which these compounds inhibit other flaviviruses, starting with other dengue virus serotypes and West Nile virus (WNV). (2) We have found that peptides from the so-called stem segment of the dengue virus E protein inhibit viral infectivity. Inhibition correlates with affinity of the peptides for the trimeric, low-pH induced conformer of the E protein ectodomain, consistent with the notion that they inhibit the zipping up step in the fusion reaction. We will determine the structures of peptide- ectodomain complexes and work out the mechanism by which these peptides inhibit entry. (3) We have developed (initially for influenza virus) a fusion assay that follows the kenetics of individual, single viron fusion events. We will use this assay to study dengue and WNV mechanisms and to correlate structure and mechanism for inhibitition of fusion by small molecules and peptides. We will also study the influence of target-membrane lipid composition on the fusion process. (4) We will develop a higher- throughput format for the single-virion fusion assay. The ultimate goal is to apply the method for screening fusion inhibitors and analyzing neutralizing antibodies.